3 research outputs found
Defective Nucleotide Release by DNA Polymerase β Mutator Variant E288K Is the Basis of Its Low Fidelity
DNA
polymerases synthesize new DNA during DNA replication and repair,
and their ability to do so faithfully is essential to maintaining
genomic integrity. DNA polymerase β (Pol β) functions
in base excision repair to fill in single-nucleotide gaps, and variants
of Pol β have been associated with cancer. Specifically, the
E288K Pol β variant has been found in colon tumors and has been
shown to display sequence-specific mutator activity. To probe the
mechanism that may underlie E288K’s loss of fidelity, a fluorescence
resonance energy transfer system that utilizes a fluorophore on the
fingers domain of Pol β and a quencher on the DNA substrate
was employed. Our results show that E288K utilizes an overall mechanism
similar to that of wild type (WT) Pol β when incorporating correct
dNTP. However, when inserting the correct dNTP, E288K exhibits a faster
rate of closing of the fingers domain combined with a slower rate
of nucleotide release compared to those of WT Pol β. We also
detect enzyme closure upon mixing with the incorrect dNTP for E288K
but not WT Pol β. Taken together, our results suggest that E288K
Pol β incorporates all dNTPs more readily than WT because of
an inherent defect that results in rapid isomerization of dNTPs within
its active site. Structural modeling implies that this inherent defect
is due to interaction of E288K with DNA, resulting in a stable closed
enzyme structure
Defective Nucleotide Release by DNA Polymerase β Mutator Variant E288K Is the Basis of Its Low Fidelity
DNA
polymerases synthesize new DNA during DNA replication and repair,
and their ability to do so faithfully is essential to maintaining
genomic integrity. DNA polymerase β (Pol β) functions
in base excision repair to fill in single-nucleotide gaps, and variants
of Pol β have been associated with cancer. Specifically, the
E288K Pol β variant has been found in colon tumors and has been
shown to display sequence-specific mutator activity. To probe the
mechanism that may underlie E288K’s loss of fidelity, a fluorescence
resonance energy transfer system that utilizes a fluorophore on the
fingers domain of Pol β and a quencher on the DNA substrate
was employed. Our results show that E288K utilizes an overall mechanism
similar to that of wild type (WT) Pol β when incorporating correct
dNTP. However, when inserting the correct dNTP, E288K exhibits a faster
rate of closing of the fingers domain combined with a slower rate
of nucleotide release compared to those of WT Pol β. We also
detect enzyme closure upon mixing with the incorrect dNTP for E288K
but not WT Pol β. Taken together, our results suggest that E288K
Pol β incorporates all dNTPs more readily than WT because of
an inherent defect that results in rapid isomerization of dNTPs within
its active site. Structural modeling implies that this inherent defect
is due to interaction of E288K with DNA, resulting in a stable closed
enzyme structure
Probing DNA Base-Dependent Leaving Group Kinetic Effects on the DNA Polymerase Transition State
We examine the DNA
polymerase β (pol β) transition
state (TS) from a leaving group pre-steady-state kinetics perspective
by measuring the rate of incorporation of dNTPs and corresponding
novel β,γ-CXY-dNTP analogues, including individual β,γ-CHF
and -CHCl diastereomers with defined stereochemistry at the bridging
carbon, during the formation of right (R) and wrong (W) base pairs.
Brønsted plots of log <i>k</i><sub>pol</sub> versus
p<i>K</i><sub>a4</sub> of the leaving group bisphosphonic
acids are used to interrogate the effects of the base identity, the
dNTP analogue leaving group basicity, and the precise configuration
of the C-X atom in <i>R</i> and <i>S</i> stereoisomers
on the rate-determining step (<i>k</i><sub>pol</sub>). The
dNTP analogues provide a range of leaving group basicity and steric
properties by virtue of monohalogen, dihalogen, or methyl substitution
at the carbon atom bridging the β,γ-bisphosphonate that
mimics the natural pyrophosphate leaving group in dNTPs. Brønsted
plot relationships with negative slopes are revealed by the data,
as was found for the dGTP and dTTP analogues, consistent with a bond-breaking
component to the TS energy. However, greater multiplicity was shown
in the linear free energy relationship, revealing an unexpected dependence
on the nucleotide base for both A and C. Strong base-dependent perturbations
that modulate TS relative to ground-state energies are likely to arise
from electrostatic effects on catalysis in the pol active site. Deviations
from a uniform linear Brønsted plot relationship are discussed
in terms of insights gained from structural features of the prechemistry
DNA polymerase active site